Pantoea trifolii sp. nov., a novel bacterium isolated from Trifolium rubens root nodules

A novel bacterium, designated strain MMK2T, was isolated from a surface-sterilised root nodule of a Trifolium rubens plant growing in south-eastern Poland. Cells were Gram negative, non-spore forming and rod shaped. The strain had the highest 16S rRNA gene sequence similarity with P. endophytica (99.4%), P. leporis (99.4%) P. rwandensis (98.8%) and P. rodasii (98.45%). Phylogenomic analysis clearly showed that strain MMK2T and an additional strain, MMK3, should reside in the genus Pantoea and that they were most closely related to P. endophytica and P. leporis. Genome comparisons showed that the novel strain shared 82.96–93.50% average nucleotide identity and 26.2–53. 2% digital DNA:DNA hybridization with closely related species. Both strains produced siderophores and were able to solubilise phosphates. The MMK2T strain was also able to produce indole-3-acetic acid. The tested strains differed in their antimicrobial activity, but both were able to inhibit the growth of Sclerotinia sclerotiorum 10Ss01. Based on the results of the phenotypic, phylogenomic, genomic and chemotaxonomic analyses, strains MMK2T and MMK3 belong to a novel species in the genus Pantoea for which the name Pantoea trifolii sp. nov. is proposed with the type strain MMK2T (= DSM 115063T = LMG 33049T).


Phenotypic and chemotaxonomic analyses
Gram stain reaction was performed using standard methods.The slides were examined using the 100 × oil immersion objective using an Olympus CX23 microscope (Olympus, Japan).The Schaeffer-Fulton staining technique was used to determine the presence of spores in the tested strains 27 .The slides were observed under the same microscope as for the Gram stain method.Growth tests were performed in YEM broth at 28 °C.The growth temperature range was tested on the same medium at 15, 28, 37, 42 and 45 °C for 4-8 days.Tolerance to NaCl was determined based on the growth of the bacteria on YEM agar supplemented with 8, 9 and 10% NaCl (w/v) concentrations and incubated 28 °C for four to eight days.The pH range for growth was established by incubating the strain in YEM broth at pH levels ranging from 5.0 to 10.0 at intervals of 1 pH unit.The Biolog GEN III system (Biolog Inc.Hayward, CA, USA) and GN A + B-ID system (Microgen) were used in accordance to the manufacturer's recommendations.Enzymatic characterization of the bacterial isolates was performed using API ZYM strips (BioMérieux, France) according to the manufacturer's protocol.Catalase and oxidase activity was determined using standard methods.Cellular fatty acids in the form of their methyl esters were prepared according to the protocol of Wollenweber and Rietschel 28 and analysed by using an Agilent Technologies (Instrument 7890) gas chromatograph connected to a mass selective detector (Agilent Technologies MSD5975C, inert XL EI/CI) (GLC-MS), using helium as a carrier.The components of fatty acid methyl ester were determined mainly by their chromatographic and mass spectral characteristics.The positions of the branching methyl group, cyclopropane ring, and the double bonds were determined by an analysis of mass spectra of fatty acid pyrrolidines 29 .Each fatty acid was quantified by calculating its peak area relative to the total peak area 30 .

In vitro assessment of plant growth promoting characteristics
Indole-3-acetic acid (IAA) production in Pantoea strains was detected using a qualitative test.The IAA production was determined using Salkowski reagent as described by Luziatelli et al. 31 .
The indole production was detected in M9 broth medium 32 with 0.5 mM of l-tryptophan as described by Gnat et al. 33 .
Assessment of HCN production by Pantoea strains was carried out by using the method described by Lorck 34 .
The ability of Pantoea strains to solubilize phosphate was determined on Pikovskaya agar 35 .The bacterial strains were streaked on Pikovskaya agar and incubated at 28 °C for 10 days.Formation of a transparent halo around the colony indicates solubilisation of phosphate.
Chrome azurol S medium (CAS, Sigma-Aldrich, USA) was used to test the capability of the microorganisms to produce siderophores 36 .The strains were spotted on CAS medium and inoculated for three to four days at 28 °C.Formation of a coloured halo around the colony indicated siderophore production.
Cellulase activity of studied strains were detected on carboxymethylcellulose (CMC) media according to the method described by Kasana et al. 37 .Gram's iodine forms a bluish-black complex with cellulose.The positive reaction for cellulase production is visible as a sharp and distinct zone around the microbial colonies 37 .
Proteolytic activity was evaluated in nutrient agar supplemented with 10% skim milk 38 .Pantoea strains were spot inoculated and incubated for 48-72 h at 28 °C.The enzymatic degradation of milk protein was visible as a clear zone around a bacterial colony.All the experiments described above were performed in triplicate.

In vitro assessment of antifungal and antibacterial activity
The antagonism test was performed against four phytopathogenic fungi listed in Table 1.Fungal strains were grown on a potato dextrose agar (PDA, Biomaxima, Poland) plates for 7 days.A 5-mm agar plug of mycelium was excised with a sterilized cork borer and was placed in the centre of a YEM agar plate inoculated with the Pantoea strain.The interactions of the tested strains with fungal pathogens were examined every day for a period of three weeks.Three biological replications were performed for each treatment.The control were plates inoculated only with the fungal pathogen.The plates were assessed as follows: (−) no effect on inhibiting the growth of the fungal phytopathogen; (+) inhibition of the growth of the fungal phytopathogen compared to the control.
The studied Pantoea strains were tested for their ability to inhibit the growth of four phytopathogenic bacteria listed in Table 1.using the agar plug diffusion method 39 .Pantoea strains were cultured on YEM agar for five days at 28 °C.After this time, 9 mm agar discs with the strain were cut out using a sterilized cork borer.An agar disc with the tested strain was placed at the centre of the LB agar plates (Biomaxima, Poland) inoculated with 24 h cultures of the phytopathogens in LB broth (Biomaxima, Poland) (10 8 CFU/ml).The plates were incubated at 28 °C and strain interactions were checked after 24, 48 and 72 h.The appearance of a zone of inhibition of the growth of phytopathogens around bacterial colonies was considered a positive result.

16S rRNA gene phylogenetic analysis
The genomic DNA was extracted using a bacterial genomic DNA extraction kit (GeneMATRIX Tissue & Bacterial DNA Purification Kit, EURx).The 16S rRNA gene was amplified and sequenced with the bacterial universal primers fD1 and rD1 described by Weisburg et al. 40 .PCR amplification reactions were carried out with ReadyMix™ Taq PCR Reaction Mix (Sigma) according to the manufacturer's recommendations.The amplified products were purified with Clean-Up purification columns (A&A Biotechnology) and sequenced with BigDye Terminator Cycle sequencing kit using the 3500 Genetic Analyzer according to the manufacturer's procedures (Life Technologies) as described elsewehere 41 .The 1337 bp long 16S rRNA gene sequence fragments of MMK2 T and MMK3 were deposited in GenBank under the accession numbers OQ799602 and OQ799603.The sequence similarity searches were performed by using the BLAST algorithm.Phylogenetic tree based on 16S rRNA gene sequences were constructed using the software MEGA X 42 and the maximum likelihood algorithm with Tamura 3-parameter model 43 .Bootstrap values were derived from 1000 replications.

Genome sequencing, annotation and phylogenomic analyses
The whole genome of strains MMK2 T and MMK3 were sequenced on a PacBio platform by Inqaba Biotechical Industries (Pty) Ltd (Pretoria, South Africa) and assembled using the SMRTLink v. 11.0 software (PacBio).Genome sequences of the type strain MMK2 T and strain MMK3 were deposited in GenBank database under the accession numbers JANIET000000000.1 and JANIES000000000.1,respectively.Genome annotation was carried out automatically with the Genome Annotation Service using the RAST tool kit 44 , available at BV-BRC web resources 45 .The antiSMASH tool was used to identify putative biosynthetic gene clusters in the analysed genomes 46 .The genomes of the type strains of all other Pantoea species were obtained from GenBank.To determine the relationship between MMK2 T , MMK3 and closely related species, phylogenomic analyses were performed using the bacterial Phylogenetic Tree Service available at BV-BRC web resources 45 and utilizing the codon tree method and the RAxML program 47 .The average nucleotide identity (ANI) and digital DNA:DNA hybridization (dDDH) relatedness between MMK2 T , MMK3 and types strains of closely related species were calculated using the ANI Calculator 48 and the Genome-to-Genome Distance Calculator 49 , respectively.

Isolation, morphology, physiology and biochemical features of Pantoea trifolii
The root nodules of legumes are inhabited mainly by bacteria capable of fixing atmospheric nitrogen as part of a symbiotic relationship with the host plant.The nitrogen-fixing symbiotic bacteria, collectively named as rhizobia, belong to different genera of Alpha-(e.g.Bradyrhizobium, Ensifer, Mesorhizobium, Rhizobium) and Betaproteobacteria (e.g.Paraburkholderia, Cupriavidus) classes 50 .In addition to the typical rhizobia, many other bacteria are often isolated from sterile root nodules, some of which are unable to fix nitrogen or induce nodulation.They are called nodule endophytes or nodule-associated bacteria 51 .Many of these nodule endophytes may be beneficial to plants due to their plant growth promoting effects 52 .

16S rRNA gene phylogeny
Using the results of the phylogenetic analysis of the 16S rRNA gene encoding sequences, we were able to estimate the taxonomic position of the studied strains at the genus level.Phylogenetic analysis based on the maximum likelihood algorithm shows that strains MMK2 T and MMK3 belong to the genus Pantoea and have the highest 16S rRNA gene similarity to P. endophytica (99.4%) and P. leporis (99.4%), followed by P. rwandensis (98.8%) and P. rodasii (98.4%), with which the studied strains clustered together, but on a distinct branch in the phylogenetic tree (Fig. 1).

Phylogenomic and genomic analyses
The phylogenomic analysis based on 500 single-copy genes found in the genomes of Pantoea sp.MMK2 T , Pantoea sp.MMK3 and reference strains clearly showed that MMK2 T and MMK3 were members of the genus Pantoea, and were most closely related to P. endophytica and P. leporis (Fig. 2).
The genome-derived ANI and dDDH values between MMK2 T , and its closely related species were between 82.96 and 93.50% (Table 4) and 26.2-53.2%(Table 5), respectively, which is all below the threshold values of 95-96% ANI and 70% dDDH, i.e. recommended cut-off values for prokaryotic species delineation 56 .
The draft genome of strain MMK2 T was 5.06 Mb long and composed of 3 contigs with a N50 of 4.16 Mb and L50 of 1 and genome coverage of 560x (Fig. 3).The genome size is above the median being 4.85 Mb for the sequenced Pantoea strains 2 .It has genomic DNA G + C content of 54.63 mol % which is within the G + C content range of the genus Pantoea 5 .The MMK2 T genome contains 4721 protein coding sequences (CDS), 78 transfer RNA (tRNA) genes, and 22 ribosomal RNA (rRNA) genes.The annotation included 733 hypothetical proteins and 3988 proteins with functional assignments (Table 6).Among the proteins with functional assignments 1241 represented proteins with Enzyme Commission (EC) numbers, 1,030 belonged to proteins with Gene Ontology (GO) assignments, and 896 included proteins that were mapped to KEGG pathways 57 .

Analysis of biosynthetic gene clusters
Analysis of the MMK2 T and MMK3 genomes using the antiSMASH tool revealed the presence of two biosynthetic gene clusters (BGC) associated with the pigments production, i.e. carotenoids and aryl polyenes (APE).The aryl polyene gene cluster showed 94% similarity to the secondary metabolite BGC from Xenorhabdus doucetiae.In addition, a cluster of genes involved in the synthesis of carotenoids was found in the genomes of studied strains.This gene cluster from strains MMK2 T and MMK3 has the classical organization crtEXYIBZ, having all the necessary carotenoid synthesis genes for zeaxanthin glucosides 58 .The role of carotenoids in bacteria is related to the protection of the cell against stress factors, such as the toxic effects of reactive oxygen species (ROS), desiccation or salinity.They also act as photoprotectants against UV radiation (especially in the range from 320 to 400 nm).It was found that carotenoids can regulate membrane fluidity and participate in the organization of membrane domains 59 .The ability to synthesise carotenoids has been described for both pathogenic and endophytic members of the genus Pantoea.They have been found in P. agglomerans, P. ananatis and in the strain Pantoea sp.YR343 [60][61][62]       No gene clusters associated with the synthesis of the blue or violet pigment were identified in the MMK2 T and MMK3 genomes.The planned research on the analysis of the structure and function of the pigment may allow the identification of genes involved in its production.In addition to the gene clusters associated with pigment production, antiSMASH found BGC involved in the synthesis of frederiksenibactin, a triscatechol siderophore, in the genomes of the studied strains.Siderophores are compounds of low molecular weight and are characterized by high affinity to metal ions, mainly ferric, but also to ions, for example, aluminium, copper, cadmium or lead 63 .In the case of plant growth promoting bacteria (PGPB), the production of siderophores can positively influence the physiological and biochemical processes taking place in the host.In addition, through competition for iron ions, they can limit the development of phytopathogens in the environment 64 .
Frederiksenibactin is produced by an opportunistic human pathogen Yersinia frederiksenii 65 but the BGC related to the synthesis of this siderophore was also identified by antiSMASH in the genomes of other Pantoea species, for example, P. endophytica, P. rodassi but not in the genome of P. rwandensis.

In vitro plant growth promoting and antimicrobial characteristics of Pantoea trifolii
Among the tested traits related to the promotion of plant growth, both studied strains showed the ability to solubilize phosphates and produce siderophores.Additionally, strain MMK2 T was positive for the production of indole-3-acetic acid (IAA).Using the methods described, we did not detect cellulolytic or proteolytic activity of either strains.The Pantoea strains tested were also negative in indole and HCN production (Table 7).Phosphate solubilization, production of siderophores and IAA appear to be plant growth-promoting traits that are widespread among Pantoea species.For example, these activities were determined experimentally for P. agglomerans C1 31 , P. eucalypti 66 , P. brenneri 67 , P. alhagi 68 and P. phytobeneficialis MSR2 9 .
Analysis of the in vitro antimicrobial activity of the MMK2 T and MMK3 strains showed that they differ in their antifungal and antibacterial properties (Table 7).It was found that the MMK2 T strain inhibits growth of Table 6.Major protein coding genes of Pantoea MMK2 T and their distribution by subsystem category as annotated by the PATRIC annotation server.www.nature.com/scientificreports/X. vesicatoria NCCB 92,059 and P. syringae pv.syringae 2905.Both MMK2 T and MMK3 strains have antifungal activity against the S. sclerotiorum strain 10Ss01.Some Pantoea strains are known to produce metabolites with antibacterial and antifungal activity, and biosynthetic gene clusters related to the biosynthesis of these compounds have been identified in their genomes 69 .No biosynthetic gene cluster associated with synthesis of antibiotics was identified in the genomes of studied Pantoea strains, suggesting another mechanism related to their antimicrobial activity.We found that the MMK2 T and MMK3 genomes contain genes encoding a complete type VI secretion system (T6SS), which can play different functions in pathogenic and non-pathogenic bacteria.One such role is to deliver effectors to neighbouring bacteria, inhibiting their growth and thus playing a role in interbacterial competition 70,71 .It was demonstrated that T6SS is a functional antibacterial system used by P. agglomerans bv.betae to deliver a lysozyme-like effector to eliminate competitors 72 .Further studies are required to determine the function of the T6SS in strains MMK2 T and MMK3.

Conclusion
Based on the results obtained from the study of phenotypic and chemotaxonomic features as well as phylogenetic and phylogenomic analyses, the MMK2 T strain should be considered a novel species of the genus Pantoea, for which we propose the name Pantoea trifolii sp.nov.The protologue description of this novel species is presented in Table 8.

Figure 2 .
Figure 2. Phylogenomic tree based on 500 single-copy genes showing the relationship between strains MMK2T, MMK3 and Pantoea type strains.Escherichia coli JCM1649 T and Enterobacter cloacae ATCC 13047 T were used as outgroups.Species names written in quotation marks are not validly published (https:// lpsn.dsmz.de/ genus/ panto ea).The branching points are labelled with bootstrap values.The bar, 0.20, represents the number of substitutions per site.The tree was generated using RAxML program.

Figure 3 .
Figure 3.A circular diagram representing the genome of Pantoea MMK2 T .The three contigs and their sizes are shown as the outermost ring.Coding sequences (CDS) on the forward strand, CDS on the reverse strand are represented by the second and third rings.The remaining rings represent RNA genes, CDS with homology to known antimicrobial resistance genes, CDS with homology to known virulence factors, guanine and cytosine (GC) content and GC skew.

Table 1 .
Bacterial and fungal strains used in in vitro antimicrobial activity tests.

Table 3 .
Cellular fatty acid profiles of MMK2 T and MMK3.a, methyl branch at the anteiso carbon atom; i, methyl branch at the iso carbon atom, n, unbranched acid; cyclopropyl, cyclopropane ring structure.

Table 7 .
In vitro plant growth promoting and antimicrobial characteristics of Pantoea sp.MMK2 T and MMK3.+ activity present; − activity absent.